tmRNA engages the bacterial ribosome in a unique process termed "trans-translation" that challenges the standard model of protein translation, in that mRNA molecules are switched during the synthesis of a single polypeptide, switching from the end of a "broken" mRNA (lacking a stop codon) to a reading frame within tmRNA. Physiological, genetic, biochemical, structural and phylogenetic approaches will be brought to bear on the broad, long-term objectives of this proposal: 1) to understand in mechanistic detail how trans-translation is accomplished, then test predictions made by the trans-translation mechanism concerning normal translation, so as to produce a unified picture of ribosome function, and 2) to understand the roles that tmRNA and trans-translation fill in the various physiological states and adaptive responses that cells undergo. Public health benefits from inhibiting bacterial infection, and biotechnological protein synthetic applications may ultimately accrue as new ways to control the ribosome emerge from the exploration of tmRNA function. Specific aims are to: 1) Identify biological roles of tmRNA in a survey of various physiological settings, evaluating benefits that tmRNA provides to the cell (using a previously developed competitive growth approach), and monitoring tmRNA expression levels. 2) Reveal structure/function relationships in the tmRNA molecule, applying previously developed methods for randomization/selection and in vivo assay of variant tmRNAs. 3) Search for factors that interact with tmRNA using genetic and biochemical approaches, identifying any complexes outside of the ribosome that include tmRNA. 4) Develop a staged in vitro trans-translation system to isolate the various complexes produced as tmRNA traverses the ribosome. 5) Analyze structure of tmRNA alone and in trans-translation complexes by chemical probing, cross-linking, crystallographic, and electron micrographic (the latter two collaborative) approaches.